Association of MDM2 Overexpression in Ameloblastomas with MDM2 Amplification and BRAFV600E Expression

Ameloblastoma is a rare tumor but represents the most common odontogenic neoplasm. It is localized in the jaws and, although it is a benign, slow-growing tumor, it has an aggressive local behavior and high recurrence rate. Therefore, alternative treatment options or complementary to surgery have been evaluated, with the most promising one among them being a targeted therapy with the v-Raf murine sarcoma viral oncogene homologue B (BRAF), as in ameloblastoma the activating mutation V600E in BRAF is common. Studies in other tumors have shown that the synchronous inhibition of BRAF and human murine double minute 2 homologue (MDM2 or HDM2) protein is more effective than BRAF monotherapy, particularly in the presence of wild type p53 (WTp53). To investigate the MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAFV600E and p53 expression. Forty-four cases of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin were examined for MDM2 overexpression and BRAFV600E and p53 expression by immunohistochemistry, and for MDM2 ploidy with fluorescence in situ hybridization. Sixteen of forty-four (36.36%) cases of ameloblastoma showed MDM2 overexpression. Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAFV600E positive and fifteen of sixteen MDM2-positive ameloblastomas (93.75%) were p53 negative. All MDM2 overexpressing tumors did not show copy number alterations for MDM2. Overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression. Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAFV600E-positive/WTp53 ameloblastomas.


Introduction
Ameloblastoma is a rare benign epithelial odontogenic neoplasm, i.e., a tumor originating from the tooth-forming epithelium.It is one of the most common odontogenic tumors and the commonest odontogenic neoplasm [1][2][3].It is usually diagnosed in patients in the third to fifth decade of life, shows a slight predilection for males, and is preferentially localized in the mandible, with the angle of the mandible and the molar region being the most commonly affected regions [3,4].Based on clinical, radiographic, and pathologic features, three types of ameloblastoma are described: the conventional solid/multicystic and the unicystic ameloblastoma are intraosseous tumors, whereas the peripheral ameloblastoma is an extraosseous tumor that develops in the gingiva.The conventional solid/multicystic ameloblastoma and the peripheral ameloblastoma are the most prevalent and the rarest In view of the complimentary roles of BRAFi and MDM2i in the treatment of various tumors, the objective of this study was to investigate MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAF V600E and p53 expression.

Results
Forty-four patients with ameloblastoma were included in this study; twenty-three were males and twenty-one females.The age range was 6-82 years and the mean age 42.6 ± 18.7 years.Thirty-five lesions were localized in the mandible and eight in the maxilla (mandible to maxilla ratio 4.3:1).Forty-one ameloblastomas were conventional solid/multicystic and three were unicystic.Solid ameloblastomas were follicular in twentyseven cases, plexiform in six cases, basaloid in six cases, and acanthomatous in two cases.Unicystic ameloblastomas were of the mural subtype, all of them showing a follicular growth pattern.Squamous metaplasia, cystic degeneration, and granular cells were seen in five, ten, and one of the follicular ameloblastomas, respectively.
Melanoma showed cytoplasmic, homogenous, and intense BRAF V600E immunostaining in most tumor cells (Figure 2A,B).Seven of sixteen MDM2-positive ameloblastomas (43.75%) were BRAF V600E positive (Figure 2C), two showed weak/ambiguous staining and were considered as negative, and seven were BRAF V600E negative.All seven positive cases were from the mandible, representing seven of ten mandibular ameloblastomas and five of seven negative cases from the maxilla, and representing five of six maxillary ameloblastomas.Almost all (14/16) were of the follicular subtype.
Table 1 shows the main clinical, histopathological, and immunohistochemical features of the 16 MDM2-positive ameloblastomas.

FISH
FISH was performed in the 16 MDM2-positive ameloblastomas.No copy number alterations for MDM2 were identified in all tumors examined (Figure 4).

FISH
FISH was performed in the 16 MDM2-positive ameloblastomas.No copy number alterations for MDM2 were identified in all tumors examined (Figure 4).

Discussion
Herein, we aimed to investigate MDM2 protein expression and gene amplification in ameloblastoma, in association with BRAF V600E and p53 expression.The main finding of the present study is that ameloblastomas overexpressing MDM2 did not show MDM2 amplification, as accessed by FISH, whereas some of them expressed BRAF V600E in the presence of WTp53, as was shown by immunohistochemistry.
In accordance with previous reports, MDM2-positive cells were seen in all cell layers [27], squamous or granular cells were MDM2 negative [31], and no difference was observed between follicular and plexiform ameloblastomas [27].Other studies have reported more intense MDM2 expression in the peripheral cells of ameloblastoma [28,29,32] and variation among the histopathological types [29][30][31][32], findings that were not confirmed in the present material.
MDM2 overexpression is considered suggestive of MDM2 amplification in various malignant tumors, such as bladder carcinoma, melanoma, and liposarcoma [51,56,57], but it has not been evaluated in ameloblastomas.In this study, all MDM2-positive cases showed MDM2 overexpression, whereas in previous studies none [28,33], 38.46% [27], or 61.54% [30] of the tumors examined showed immunohistochemical expression consistent with overexpression.However, none of the 16 MDM2-overexpressing tumors showed MDM2 amplification by FISH.Those findings are in line with previous studies [10,12,13,60,61] that by employing various molecular techniques, e.g., microarrays, RNA-sequencing, Sanger sequencing, and polymerase chain reaction (PCR), highlighted the differential expression and/or mutations of other molecular markers that do not predict MDM2 mutations in ameloblastoma.
MDM2 transcription may be activated by binding on its P2 promoter, located in the first intron of the molecule, by Activator protein 1 (AP-1) and E26 transformationspecific or E-twenty-six (ETS) transcription factors that are downstream molecules of the BRAF pathway [34].The MAPK pathway may be constitutively activated by BRAF V600E mutation [51] that is frequently detected in ameloblastomas [10][11][12][13][14][15]17].Seven of sixteen MDM2-positive cases in the present study were shown by immunohistochemistry to be positive for BRAF V600E (43.75%), compared with the reported 46% to 82% positivity for this marker in other studies [10][11][12][13][14][15]17], and most of them were in the mandible [10,17].Although the gold standards for detecting BRAF V600E mutation are PCR and DNA sequencing, immunohistochemistry with VE1 antibody, as applied in the present study, shows high concordance with molecular techniques [12,70].TGF-β upregulates MDM2 expression via the interaction of Smad2 and Smad3 with the P2 promoter located in the first intron of MDM2 [34].In ameloblastomas, low expression of TGF-β1 and functional pSmad2/3 and Smad4 proteins do not indicate a critical role for the TGF-β pathway [71].Those findings support the suggestion that MAPK activation through BRAF V600E mutation may be the main cause of MDM2 overexpression in BRAF V600E -positive tumors.As for BRAF V600E -negative cases that overexpress MDM2, it should be noticed that MAPKactivating mutations other than BRAF V600E have been identified in ameloblastomas [16], and in some ameloblastomas the TGF-β pathway may be activated [71].Further evaluation of MDM2 overexpression/MAPK activation association could show its possible utility as a marker of MAPK activation.
In cutaneous melanomas that overexpress MDM2 without MDM2 amplification and which have normal p53, inhibition of MDM2 may reconstitute WTp53 action in tumor cells and suppress tumor growth [52].Furthermore, MDM2i may act as a complement to BRAFi in malignant neoplasms that overexpress MDM2, are BRAF V600E positive, and express WTp53.Dual BRAF/MDM2 inhibition suppressed the viability of WTp53 melanoma cells in vitro and WTp53 melanoma growth in vivo [54], and in cell cultures of cutaneous melanoma [74][75][76] and colon carcinoma [77] this led to restoration of p53 function, possibly promoting apoptosis and suppressing tumor growth.In mice xenografted with RKO colon carcinoma cell inhibitors of BRAF and MDM4, a nuclear protein structurally homologous to MDM2 that interacts with both p53 and MDM2, this treatment managed to shrink the tumor by 80%, when the response to each drug tested separately was 23% and 24%, respectively [77].Furthermore, dual inhibition helped overrun tolerance to BRAFi, an adverse effect attributed to reactivation of the MAPK pathway [22,53,54].In BRAF V600Epositive ameloblastomas which were unresectable due to multiple recurrences and lung metastases, monotherapy with BRAFi dabrafenib [19,21] or vemurafenib [20], neoadjuvant treatment with dabrafenib [24][25][26], or dual BRAF/MEK inhibition with dabrafenib and trametinib [18,22,23,26] showed good response without severe toxicity.The addition of MDM2i such as nutlins, which disrupt the MDM2-p53 interaction by competing with p53 for binding to the MDM2 protein, could augment the therapeutic outcome and possibly overcome tolerance to BRAFi [23].
A limitation of the present study is that BRAF V600E and p53 expression were not examined in MDM2-negative ameloblastomas of our sample, as the investigation was focused on MDM2-overexpressing ameloblastomas.Therefore, the association of BRAF V600E and p53 expression with MDM2 status cannot be concluded, although for the latter it is expected, based on the available literature, that the tumors would be p53 negative.

Materials and Methods
The cohort consisted of 44 biopsies of ameloblastoma fixed in 10% buffered formalin and embedded in paraffin (FFPE).Histopathologic diagnosis in each case was confirmed by microscopic examination of 5 µm thick FFPE tissue sections stained with hematoxylin and eosin by all researchers according to the World Health Organization diagnostic criteria for ameloblastoma [5].Relevant clinical information was retrieved from the pathology request forms, tabulated, and anonymized.The study was approved by the Ethics Committee of the Dental School, National and Kapodistrian University of Athens (#302), and the Institutional Review Board of the University of Minnesota (IRB #1604E86681), and was conducted in accordance with the principles of the Declaration of Helsinki.
Positive controls were sections from two cases of atypical lipomatous tumor of the thigh for MDM2, two cases of oral mucosa for p53, and one case of BRAF V600E -positive cutaneous melanoma for VE1.For negative controls, the primary antibodies were substituted with Negative Control-monoclonal (Ventana, Medical Systems Inc., Tucson, AZ, USA).

FISH
FISH was performed on 5 µm thick FFPE tissue sections with the commercially available ZytoLight-FISH tissue implementation kit and ZytoLight-FISH SPEC MDM2/CEN12 Dual Color Probe (ZytoVision®GmbH, Bremerhaven, Germany).This is a direct labeling technique optimized for use with FFPE tissue sections, with ready-to-use fluorescencelabeled polynucleotide probes: a green one targeting the chromosomal region of the human MDM2, and an orange one targeting DNA sequences of centromeric alpha-satellites of chromosome 12 (CEN12).Alpha-satellite sequences of CEN12 served as an internal control and as a measure for DNA integrity.Sections were examined with an oil-immersion ×100 lens and proper fluorescence filters (green-labeled polynucleotides: excitation at 503 nm and emission at 528 nm, orange-labeled polynucleotides: excitation at 547 nm and emission at 572 nm).Amplification of the MDM2 gene locus was defined as an MDM2/CEN12 signal ratio ≥2 in >10% of the total number of cells or as clustering of multiple copies of green signals [79,80].In ameloblastomas, CEN12 was expected to be euploid, as chromosomal copy number variations for this tumor are rare and do not include chromosome 12 [73,[81][82][83][84].Therefore, no external controls were necessary.Fifty interphase nuclei from different areas of the FISH slides were evaluated in each case.

Statistical Analysis
Statistical analysis was performed with the SPSS, v25.0 Software for Windows (SPSS Inc., Chicago, IL, USA).Associations between the MDM2 immunohistochemistry results and patients' demographic characteristics were investigated with the Chi Square Test and, when expected frequency was <5, with the Fisher Exact Test.The level of statistical significance was set at p-value (p) < 0.05.

Conclusions
In conclusion, overexpression of MDM2 in ameloblastomas is not associated with MDM2 amplification, but most probably with MAPK activation and WTp53 expression.Further verification of those findings could form the basis for the use of MDM2 expression as a marker of MAPK activation in ameloblastomas and the trial of dual BRAF/MDM2 inhibition in the management of MDM2-overexpressing/BRAF V600E -positive/WTp53 ameloblastomas.

Figure 4 .
Figure 4. Fluorescence in situ hybridization (FISH) for MDM2 in a follicular ameloblastoma (ZytoLight-FISH tissue implementation kit).The orange signals represent the probe and the green the control probe.The presence of two orange and two green hybridized signals is representative of euploidy.

Figure 4 .
Figure 4. Fluorescence in situ hybridization (FISH) for MDM2 in a follicular ameloblastoma (ZytoLight-FISH tissue implementation kit).The orange signals represent the probe and the green the control probe.The presence of two orange and two green hybridized signals is representative of euploidy.